Microrespirometer and associated methods

ABSTRACT

A method for measuring an evolution rate of a gas from a sample includes equilibrating a sample with an alkaline solution and a pH indicator and permitting the alkaline solution to absorb formed carbon dioxide in an enclosed headspace. From the pH indicator at equilibrium is determined a time increment at which an increment of the alkaline solution is consumed by the CO 2 . Carbon dioxide evolution rate is calculated from the time increment, the volume increment, and the alkaline solution concentration. A device for performing this measurement includes a sample vial and a reaction chamber having an opening adapted for mating with a sample vial opening and an opening for receiving the solution. The reaction chamber is dimensioned for equilibrating the sample with the alkaline solution and for determining the time increment required for an increment of the alkaline solution to be consumed by CO 2 .

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from commonly owned provisional application Ser. No. 60/249,771, filed Nov. 17, 2000, “Carbon Dioxide Microrespirometer.”

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a device and method for determining gas evolution rates in solids and liquids, and, more particularly, to such a device and method for determining carbon dioxide evolution rates in a sample.

[0004] 2. Description of Related Art

[0005] Respiration is a common indicator of biological activity. Respirometry, the measurement of respiration rates, has been applied to a broad spectrum of applied and environmental microbiology, such as toxicity, with treatability, process control, and prediction of biological oxygen demand (BOD₅) in wastewater treatment, assessment of metal toxicity, living soil microbial biomass, and food quality.

[0006] Respiration rates can be measured either by rates of oxygen consumption or CO₂ evolution. Rapid oxygen consumption rate can be measured by using an oxygen probe or a quantitative electrolytic cell. Most oxygen respirometers, however, are applicable only to liquid samples. Oxygen respirometers with an electrolytic cell can be used to determine respiration of solid or semisolid samples, but their sensitivity is compromised.

[0007] Sensitive and rapid CO₂ respirometers based on infrared (ir) detectors have been developed in the past three decades and can handle solid samples with high speed and sensitivity. Instrumental respirometers are technically complicated and expensive if accuracy and sensitivity are needed. Noninstrumental CO₂ respirometers operated by an alkaline trap and acid-base titration have been in existence for many years. They are simple but relatively slow, with a measurement time in days, and less sensitive, with a detection limit in mL CO₂/day. Sensitive and rapid determination of respiration rates is highly desirable in monitoring microbial activity in food and environmental samples. A desired sensitivity, for example, would comprise one in the microliter CO₂ per hour level, and a rapidity of determination within about an hour.

SUMMARY OF THE INVENTION

[0008] It is therefore an object of the present invention to provide a device and method for determining gas evolution rates rapidly and sensitively.

[0009] It is another object to provide such a device and method for determining CO₂ evolution rates directly.

[0010] It is an additional object to provide such a device and method for use with solid or liquid samples.

[0011] It is a further object to provide such a device and method having a modest cost.

[0012] It is also an object to provide such a device and method operable under laboratory or remote site conditions.

[0013] These and other objects are attained by the present invention, a first aspect of which is a method for measuring an evolution rate of a gas from a sample. The method comprises the steps of pre-equilibrating a sample with a solution comprising an alkaline solution and a pH indicator and permitting the alkaline solution to absorb formed carbon dioxide in an enclosed headspace. After the CO₂ absorption/evolution equilibrium is attained, from a change in the pH indicator is determined a time increment at which a small increment of the alkaline solution is substantially consumed by the CO₂ evolved. A calculation is made of a carbon dioxide evolution rate from the time increment, the small increment volume and concentration of the alkaline solution.

[0014] Another aspect of the invention is a device for measuring an evolution rate of a gas from a sample. The device comprises a sample vial having an opening into an interior space for containing a sample therein. The device further comprises a reaction chamber having an opening adapted for mating with the sample vial opening and a solution-receiving opening for receiving a solution comprising an alkaline solution and a pH indicator. The reaction chamber is dimensioned for receiving a predetermined amount of the alkaline solution to absorb formed CO₂ from a sample within the headspace.

[0015] A further aspect of the invention is a system for measuring an evolution rate of a gas from a sample. The system comprises a respirometer device as described above and means for determining from a change in color in the pH indicator a time increment at which a small increment of the alkaline solution is substantially consumed by the CO₂ from the sample.

[0016] The features that characterize the invention, both as to organization and method of operation, together with further objects and advantages thereof, will be better understood from the following description used in conjunction with the accompanying drawing. It is to be expressly understood that the drawing is for the purpose of illustration and description and is not intended as a definition of the limits of the invention. These and other objects attained, and advantages offered, by the present invention will become more fully apparent as the description that now follows is read in conjunction with the accompanying drawing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a schematic illustration of a microrespirometer of the present invention.

[0018]FIG. 2 is a graph of CO₂ absorption versus shaking rate of the microrespirometer.

[0019]FIG. 3 is a graph of CO₂ absorption and concentration of an alkaline solution in the microrespirometer.

[0020]FIG. 4 is a graph of CO₂ absorption rate versus CO₂ concentration in the headspace of the microrespirometer.

[0021]FIG. 5 is a graph of the percent of equilibrated value versus pre-equilibration time in the microrespirometer for a range of evolution rates

[0022]FIG. 6 is a graph of CO₂ evolution rate versus the rate determined by the microrespirometer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] A description of the preferred embodiments of the present invention will now be presented with reference to FIGS. 1-6.

[0024] The basis of the system 10 and method of the present invention is to establish a carbon dioxide absorption/evolution equilibrium between an alkaline solution and a sample. After the equilibrium is attained, an indicator comprising, for example, phenolphthalein, is used to indicate the end point of a small increment of the alkaline solution being consumed by the CO₂ evolved.

[0025] The system 10 of the present invention comprises a microrespirometer device 11 (FIG. 1), which in turn comprises a substantially transparent reaction chamber 12 and sample vial 13. The reaction chamber 12 comprises a small alkaline trap with a total headspace of 6-7 mL having a small septum hole 14. The sample vial 13 size is variable, and exemplary sizes include 25, 30, 40, and 75 mL (e.g., Fisherbrand EPA bottles, Suwanee, Ga.). The reaction chamber 12 and sample vial 13 are coupled through a standard threaded screw 15 and septum liner 16 to form a closed headspace 17.

[0026] An alkaline solution 21 is injectable, such as using a syringe 18, into the reaction chamber 12 via a solution-receiving opening 18, and a sample 19 is placeable in the sample vial 13. The alkaline solution absorbs the CO₂ in the headspace 17. The indicator in the alkaline solution changes color when the alkaline solution is “consumed” by CO₂. Preferably the microrespirometer 11 is shaken at a fixed rate (e.g., 240 rpm) on an orbital shaker 20 to enhance CO₂ absorption.

[0027] The alkaline solution of the present invention comprises a solution of NaOH, BaCl₂, and indicator, with an equal molar ratio of NaOH and BaCl₂ and 0.5 mL indicator solution, with 0.5% phenolphthalein in 50% ethanol solution, per 50 mL alkaline solution. BaCl₂ in the alkaline solution precipitates the absorbed CO₂, which ensures the stoichiometry of 2 moles of alkaline spent per mole of CO₂ absorbed:

CO₂+2NaOH+BaCl₂=BaCO₃(s)+2NaCl   (1)

[0028] BaCl₂ also sharpens the change of color at the end point when a very low level of respiration is being determined. The alkaline solution is stored in a septum-capped vial to prevent absorption of CO₂ from the air. The alkaline solution is transferred through, for example, a syringe 18.

[0029] Optimal operating conditions for the system 10 were determined with a series of experiments. The effect of shaking on the CO₂ absorption of the microrespirometer 11 was investigated by coupling microrespirometers 11 with empty 25-mL sample vials 13 in a glove box having a known CO₂ concentration, as determined with an ir CO₂ analyzer.

[0030] A 0.2-mL portion of 0.002 M alkaline solution was injected into each reaction chamber 12. The microrespirometers 11 were shaken at fixed rates of 100, 150, 200, 250, and 300 rpm. The time required to consume the alkaline solution in each microrespirometer 11, as indicated by the indicator color change, was recorded. Each test was repeated in triplicate, and the results are plotted in FIG. 2. The CO₂ absorption is shown to increase as the shaking rate is increased from 100 to 250 rpm. The increase in CO₂ absorption levels off when the shaking rate exceeded 250 rpm. Shaking at 200 rpm or higher improves reproducibility of CO₂ absorption. A fixed shaking rate between 200 and 250 rpm is recommended for the microrespirometer 11 because the benefit of shaking is achieved while the difficulty of operation at higher rates is avoided.

[0031] The effect of alkaline concentration on the absorption of CO₂ in a closed headspace 17 was investigated at 25° C. A 25-mL sample vial was connected to an ir analyzer so that the vial 13 and the ir detector formed a closed headspace 17 in which air circulated continuously. The 25-mL vial 13 was shaken at 240 rmp on an orbital shaker 20. 1-mL portions of 0.2, 0.1, 0.01, and 0.0011 M were injected into the vial 13 through the solution-receiving opening 18 at the beginning of the experiment, and the concentration of CO₂ in the vial 13 was recorded periodically.

[0032] The experiment was repeated twice, and the results are plotted in FIG. 3, where each dot represents a single measurement. It can be seen that as the concentration of alkaline solution decreases from 0.2 to 0.01 M, the CO₂ absorption rate decreases as well. The CO₂ absorption rate does not decrease further as the alkaline concentration is reduced from 0.01 to 0.001 M. It is not believed possible to have complete absorption of CO2 in the headspace 17 of the microrespirometer 11 in a matter of hours when the concentration of the alkaline solution is less than 0.01 M. The concentration of the alkaline solution has to be much less than 0.01 M in order to determine CO₂ evolution rate at a microliter per hour level. The microrespirometer 11 therefore does not work on the principle of complete CO₂ absorption, but on an absorption/evolution equilibrium principle that will be discussed in the following.

[0033] An alkaline solution of less than 0.0005 M is not sufficiently stable to be used in the microrespirometer 11 because the possibility of contamination from ambient CO₂ is too large for such low alkalinity. Phenolphthalein is not stable in alkaline concentrations exceeding 0.01 M; the deep pink color fades away by itself within 1 h. Therefore, a preferred alkaline concentration range suitable for the microrespirometer 11 is between 0.01 and 0.001 M.

[0034] The relationship between CO₂ absorption rate and the CO₂ concentration in the headspace 17 of the microrespirometer 11 was also investigated. Microrespirometers 11 with a 75-mL sample vial 13 were coupled in a glove box of known CO₂ concentration. Increments of 0.1 mL 0.002 M alkaline solution were injected into the reaction chamber 12. The microrespirometers 11 were shaken at 240 rpm, and the time required to consume each increment of the alkaline solution was recorded. The consumption of each increment of the alkaline solution, for example, 0.2 μmol alkaline, or 0.1 μmol CO₂, represents a 29.7-ppm (v/v) reduction of CO₂ concentration in the 82-mL microrespirometer 11 at 25° C. Each treatment was performed in triplicate, and the results are plotted in FIG. 4, with each dot representing a single measurement.

[0035] In using the microrespirometer 11 of the present invention, a portion of solid or liquid sample 19 is placed in the sample vial 13, and the vial 13 is coupled to the reaction chamber 12. 0.8 mL alkaline solution of a desired concentration is injected into the reaction chamber 12 using a syringe 18. The respirometer 11 is shaken at a fixed rate, for example, 240 rpm, for 30 min, which comprises the pre-equilibration period, ensuring that the alkaline solution is not completely consumed during this time. If the alkaline solution is about to be consumed, more alkaline solution is injected into the reaction chamber 12. After the 30-min pre-equilibration the shaker 20 is stopped, and the alkaline solution in the chamber 12 is withdrawn to leave 0.1-0.2 mL. The respirometer 11 is continued to be shaken until the alkaline solution changes to a faint pink color. The shaker 20 is stopped immediately, and 0.1 mL alkaline solution is injected, shaking is resumed, and the time required to consume the alkalinity is recorded.

[0036] In an alternate embodiment, all the alkaline solution in the chamber 12 is withdrawn, and a new 0.1 mL portion of alkaline solution is injected prior to resuming the shaking.

[0037] In either case, once the first indicator change has been recorded, increments of 0.1 mL alkaline solution are injected a predetermined number of further times, for example, twice more, and the time required to consume each increment is recorded.

[0038] The average of the times required to consume each 0.1-mL increment is used to calculate CO₂ evolution rate using the following formula:

carbon dioxide evolution rate (μmol/h)=(0.1×10³ ×M/2)/60t  (2)

[0039] where M is the concentration of the alkaline solution in mol/L and t is the time required to consume the 0.1-mL increment in min. The CO₂ evolution rate can be expressed in microliters per hour by multiplying the molar volume of CO₂ at a specific temperature.

[0040] The relationships between the CO₂ absorption rate of a 0.002 Malkaline solution and the concentration of the CO₂ in the headspace 17 is shown in FIG. 4. In general, the CO₂ absorption rate has a positive curve-linear relationship with the concentration of CO₂. The absorption rate of the respirometer 11 at a given temperature and shaking rate reflects the CO₂ concentration in the headspace 17, which may not be the CO₂ evolution rate of the sample. However, if a sample is equilibrated with the alkaline solution in the respirometer at a given temperature and shaking rate, the concentration of CO₂ in the respirometer would eventually reach a constant value when the CO₂ absorption rate equals the CO₂ evolution rate. For example, if the starting CO₂ evolution rate of the sample 19 is 100 μL/h, the CO₂ concentration of the respirometer 11 is increased to about 660 ppm and remain there because an equilibrium of CO₂ absorption and evolution is established. If the CO₂ evolution rate of the sample is 20 μL/h, the CO₂ concentration of the respirometer 11 is decreased to about 150 ppm, where an absorption/evolution equilibrium is established. The CO₂ evolution rate of a sample 19, therefore, can be determined by the CO₂ absorption rate of the microrespirometer 11 when an equilibrium or steady state is established. That is, after a sample is equilibrated with an alkaline solution in a microrespirometer 11 of the present invention, the CO₂ evolution rate can be determined by the time required to consume a small increment of the alkaline solution, as shown in Eq. (2).

[0041] The minimum time required for a sample 19 in the respirometer 11 to reach an equilibrium is deduced from a computer simulation based on a relationship between the CO₂ absorption rate and the CO₂ concentration of the respirometer 11 and the CO₂ evolution rate of the sample 19. That is, the concentration of CO₂ in the headspace 17 after being shaken for a small increment of time Δt is

C _(i+Δt) =C _(i)+(E−A _(Ci))Δt/V _(headspace)   (3)

[0042] where C_(i) and C_(i+t) are the CO₂ concentrations of the respirometer at time i and time i+Δt, respectively. A_(Ci) is the CO₂ absorption rate of the respirometer at time i and is a function of the CO₂ concentration C_(i). E is the CO₂ evolution rate of the sample 19, and V_(headspace) is the volume of the headspace 17.

[0043] The mathematical relationship of A_(Ci) and C_(i) was generated by a nonlinear regression curve fitting program (Table Curve, Jandel Scientific, San Rafael, Calif.) using the data of FIG. 4. The regression enabled the calculation of A_(Ci) based on C_(i). The values of A_(Ci), C_(i), and C_(i+Δt), for each small time increment (0.5 min) of Δt were calculated and tabulated using a spreadsheet software (Excel, Microsoft, Redmond, Wash.) based on Eq. (3). An equilibrium is attained in the simulation when the CO₂ concentration in the respirometer approaches a constant, i.e., (E−A_(Ci)) approaches 0 and C_(i+t) approaches C_(i). The minimum time required to attain an equilibrium is the sum of all small time increments, Δt, during which CO₂ concentration approaches a constant. The ratio of the CO₂ absorption rate to evolution rate (i.e., A_(Ci)/E) expressed as a percentage of the CO₂ evolution rate during the time source of reaching an equilibrium is presented in FIG. 5. Two headspace volumes of the respirometer, i.e., 12 mL (5 mL remaining headspace in the sample vial plus 7 mL in the reaction chamber) and 27 mL (20 mL remaining headspace in the sample vial plus 7 mL in the reaction chamber) were simulated in FIG. 5.

[0044] The results indicate that the smaller the headspace 17, the quicker an equilibrium is reached, and that the greater the CO₂ evolution rates, the quicker an equilibrium is reached. For example, in the 12-mL headspace case, 30 min pre-equilibration is sufficient for the measurement of all CO₂ evolution rates ≳1 μL/h. In the 27 mL headspace case, 100-107% of equilibrated value can be attained within 45 min for all CO₂ evolution rates, except the 1 μL/h case. The working range of the respirometer is designed to be 1-300 μL/h, which requires 30-45 min of pre-equilibration time, according to the condition of this study, to measure accurately the CO₂ evolution rate. If the CO₂ evolution rate is very low (≳5 μL/h), the headspace 17 of the respirometer 11 should be kept minimal to hasten the equilibration. The respirometer 11 was designed so that the size of the reaction chamber 12 stays the same while the size of the sample vial 13 may vary according to the need of samples and the requirement of a minimal headspace 17.

[0045] A validation experiment was performed by comparing results using the microrespirometer 11 with a method using an ir analyzer such as known in the art. Portions of soil samples of relatively low CO₂ evolution rates (2-5 μL/h/g), unfrozen processed meat samples of medium CO₂ evolution rates (10-100 μL/h/5 g), and room-temperature milk samples of high CO₂ evolution rates (80-280 μL/h/20 mL) were placed in 25-mL sample vials 13. The CO₂ evolved by microorganisms associated with each sample was determined by the microrespirometer 11 method of the present invention. A duplicate sample in another 25-mL sample vial 12 was also placed in a 250-mL flask, and the CO₂ evolution rate was determined by the ir analyzer method known in the art. The sample vials 12 in the microrespirometers 11 and those in the 250-mL flasks of the ir analysis method were exchanged, and the CO₂ evolution rates determined again with the alternate methods.

[0046] One of the advantages of the microrespirometer 11 is its ability to determine the CO₂ evolution rate accurately at the μL/h level in a short time. Determination of the CO₂ evolution rates at a μL/h level is quite a challenge even for a sophisticated ir method. The IR analyzer must be able to detect less than 10 ppm (v/v) changes of CO₂ concentration with certainty during a period of hours. The accuracy of an IR analyzer method is further limited by the uncertainty of the volume occupied by a solid sample, and, therefore, that of the headspace, in most cases. Variation of headspace humidity, pressure, and temperature all affect the accuracy and precision of an ir respirometer. Because the microrespirometer method is based on the principle of CO₂ absorption-evolution equilibrium, its accuracy is not affected by headspace volume, humidity, pressure, or initial CO₂ concentration. The simplicity, noninstrumental nature, and very modest costs of the microrespirometer 11 make it available to many laboratory and field applications where accurate and rapid determination of respiration rate is desired.

[0047] In the foregoing description, certain terms have been used for brevity, clarity, and understanding, but no unnecessary limitations are to be implied therefrom beyond the requirements of the prior art, because such words are used for description purposes herein and are intended to be broadly construed. Moreover, the embodiments of the apparatus illustrated and described herein are by way of example, and the scope of the invention is not limited to the exact details of construction.

[0048] Having now described the invention, the construction, the operation and use of preferred embodiment thereof, and the advantageous new and useful results obtained thereby, the new and useful constructions, and reasonable mechanical equivalents thereof obvious to those skilled in the art, are set forth in the appended claims. 

What is claimed is:
 1. A method for measuring an evolution rate of a gas from a sample, the method comprising the steps of: equilibrating a sample with a solution comprising an alkaline solution and a pH indicator; permitting the alkaline solution to absorb formed carbon dioxide in an enclosed space; following the equilibrating step, determining from a change in the pH indicator a time increment at which an increment of the alkaline solution is substantially consumed by the carbon dioxide; calculating from the time increment a carbon dioxide evolution rate.
 2. The method recited in claim 1, wherein the equilibrating step comprises shaking the sample and the solution to enhance carbon dioxide absorption.
 3. The method recited in claim 1, wherein the shaking step comprises shaking at a fixed rate.
 4. The method recited in claim 1, wherein the alkaline solution comprises sodium hydroxide and barium chloride.
 5. The method recited in claim 1, wherein the indicator comprises phenolphthalein.
 6. The method recited in claim 5, wherein the indicator further comprises an ethanol solution.
 7. The method recited in claim 1, further comprising the step of performing a pre-equilibration comprising the steps, prior to the equilibrating step, of: equilibrating the sample with a first amount of the solution comprising an alkaline solution and a pH indicator, the first amount sufficient to absorb the carbon dioxide formed during a predetermined amount of time; permitting the alkaline solution to absorb the formed carbon dioxide in the enclosed space for the predetermined amount of time; and withdrawing the alkaline solution to leave a predetermined portion in the reaction chamber following the equilibrating step.
 8. The method recited in claim 7, wherein the portion comprises substantially none of the solution.
 9. The method recited in claim 7, wherein the equilibrating step comprises equilibrating a sample with a predetermined quantity of the alkaline solution and the portion comprises the predetermined quantity.
 10. The method recited in claim 1, wherein the equilibrating step comprises injecting a predetermined quantity of the alkaline solution into the reaction chamber.
 11. The method recited in claim 10, further comprising the steps of: repeating the equilibrating, permitting, and determining step a predetermined number of times; and averaging the time increments from the repeated equilibrating, permitting, and determining steps; and wherein the calculating step comprises calculating from the averaged time increment a carbon dioxide evolution rate.
 12. The method recited in claim 1, wherein the change in the pH indicator comprises a visualizable color change.
 13. The method recited in claim 1, wherein the calculating step comprises using the following equation: carbon dioxide evolution rate=(0.1×103×M/2)/60t, wherein M is the concentration of the solution and t is the time increment.
 14. A device for measuring an evolution rate of a gas from a sample, the device comprising: a sample vial having an opening into an interior space for containing a sample therein; and a reaction chamber having an opening adapted for mating with the sample vial opening and a solution-receiving opening for receiving a solution comprising an alkaline solution and a pH indicator, the reaction chamber dimensioned forequilibrating the sample with a predetermined amount of the solution to attain a CO₂ absorption/evolution equilibrium between the alkaline solution and the sample.
 15. The device recited in claim 14, wherein the sample vial has a threaded coupling adjacent the opening and the reaction chamber has a septum liner leading to the sample vial, the septum liner matable with the threaded coupling.
 16. The device recited in claim 14, wherein the reaction chamber comprises a substantially transparent spherical member and the solution-receiving opening is adapted for receiving a syringe tip thereinto.
 17. A system for measuring an evolution rate of a gas from a sample, the system comprising: a respirometer device comprising: a sample vial having an opening into an interior space for containing a sample therein; and a reaction chamber having a mixing opening adapted for mating with the sample vial opening and a solution-receiving opening for receiving a solution comprising an alkaline solution and a pH indicator, the reaction chamber dimensioned for equilibrating the sample with a predetermined amount of the alkaline solution; and means for determining from a change in the pH indicator a time increment at which an increment of the alkaline solution is substantially consumed by the formed CO₂.
 18. The system recited in claim 17, further comprising means for shaking the sample and the solution to enhance carbon dioxide absorption.
 19. The system recited in claim 18, wherein the shaking means comprises means for shaking at a fixed rate.
 20. The system recited in claim 19, wherein the shaking means comprises an orbital shaker.
 21. The system recited in claim 17, further comprising a syringe for injecting solution into the reaction chamber.
 22. The system recited in claim 17, wherein the alkaline solution comprises sodium hydroxide and barium chloride.
 23. The system recited in claim 17, wherein the indicator comprises phenolphthalein.
 24. The system recited in claim 23, wherein the indicator further comprises an ethanol solution.
 25. The system recited in claim 17, further comprising means for withdrawing at least some of the solution following a pre-equilibration period to leave a predetermined portion in the reaction chamber. 